Solubilization of flavonols

ABSTRACT

A method of increasing the solubility in water of a flavonol component of a flavonol-containing composition, the method including the steps of providing a flavonol-containing composition, providing an anthocyanin-containing composition and mixing the two compositions.

This Application is a continuation-in-part of U.S. application Ser. No.08/978,158, filed Nov. 25, 1997, now U.S. Pat. No. 6,086,910, which is acontinuation-in-part of U.S. application Ser. No. 08/934,055 filed Sep.19, 1997, now U.S. Pat. No. 6,099,854.

FIELD OF THE INVENTION

This invention relates to a method of increasing the solubility offlavonols and their related compounds in water and aqueous solutions,and to a method of providing soluble flavonol-containing compositions.

BACKGROUND TO THE INVENTION

Polyphenols are those compounds which comprise more than one phenolicgroup. Among the polyphenols are the following classes: flavonoids (aterm often used to denote polyphenols in general, but more commonly inEurope to denote only the flavones), the flavanols, proanthocyanidins(also called procyanidols, procyanins, procyanidins and tannins) andanthocyanins.

The flavones are compounds with a basic structure shown in FIG. 1 inwhich two benzene rings (A and B) are linked with a heterocyclic sixmember ring C containing a carbonyl group. Ring B can be joined inposition 2 (as illustrated) to give a flavone or to position 3 to givean iso flavone. Hydroxylation can occur at one or more of positions 3,5, 7, and 3′, 4′, 5′ to give compounds called flavonols. Typicalexamples of flavonols are: quercetin (hydroxylated at positions 3, 5, 7,3′, 4′), kaempferol (hydroxylated at positions 3, 5, 7, 4′), andmyricetin (hydroxylated at positions 3, 5, 7, 3′, 4′, 5′). Flavonols canexist naturally as the aglycone or as O-glycosides (e.g. with D-glucose,galactose, arabinose, rhamnose etc). Other forms of substitution such asmethylation, sulphation and malonylation are also found.

The flavanols have a basic structure shown in FIG. 2. The two mostcommon flavanols are catechin (hydroxyl groups at positions 5, 7, 3′,4′) and its stereo-isomer epi-catechin. The hydroxyl groups can beesterified with gallic acid. The proanthocyanidins are polymers ofcatechin and/or epicatechin and can contain up to 8 or more repeatunits. These compounds are often called proanthocyanidins, procyanidinsor tannins.

The anthocyanins are colored substances, sometimes calledanthocyanidins. The monomeric anthocyanins have a basic structure asshown in FIG. 3. Typical examples are: cyanidin (hydroxylated atpositions 3, 5, 7, 3′, 4′), delphinidin (hydroxylated at positions 3, 5,7, 4′, 5′) and pelargonidin (hydroxylated at positions 3, 5, 7, 3′). Thehydroxyl groups are usually glycosylated and/or methoxylated (e.g.malvidin is substituted at the 3′ and 5′ hydroxyl groups and paeonidinand petunidin are substituted at the 3′ hydroxyl group). In additionpolymers of these anthocyanins exist which are classified as polymericanthocyanins.

Within the general term “polyphenols” are also included the dihydroxyand trihydroxy benzoic acids and the phytoalexins, a typical example ofwhich is resveratrol.

Polyphenols are found in various amounts in large numbers of naturalproducts especially plant material such as fruit and vegetables. Aparticular rich source is the grape, in which the polyphenols areplentiful in the skins and seeds, but not in the pulp. During themanufacture of grape juice, quantities of polyphenols are expressed intothe juice, and the polyphenol content will depend on such factors as thetype of grape, the climate in which it is grown, and the manufacturingprocess used in making the juice. Some grape juice, especially that madefrom the Concord grape, may contain as much as 2.5 g polyphenol perlitre of juice. Grape skins and seeds are commercially extracted withwater and other solvents to obtain polyphenols. In addition, polyphenolsfrom grape skins and seeds become incorporated into wine during thevinification process. Red wine is made by maintaining contact betweenthe fermenting liquor and the crushed grape residue (pomace) forprolonged periods, whilst in the manufacture of white wine the grapeskins are removed relatively quickly. Accordingly, wine in general, andred wine in particular, contains reasonable amounts of polyphenols,amounting to about 1-3 g/L and is thus a potential commercial source ofpolyphenolic compounds.

Polyphenols are known to have antioxidant properties and have potentialuse in the food, cosmetic and pharmaceutical industries. Among thepolyphenols, the flavonols have been shown to have many usefulproperties as antioxidants, and to decrease platelet stickiness.

Epidemiological studies have shown that countries and people with a highflavonol intake have less coronary heart disease (Hertog et al, 1995Arch. Int. Med. 155, 381-6).

In unprocessed fruit and vegetables the flavonols occur as glycosidesand the aglycone is absent. The most abundant flavonol is quercetin.Among vegetables the highest concentration of quercetin glycosides is inonions (3 to 500 mg/kg), kale (100 mg/kg) French beans (30 to 45 mg/kg),and broccoli (30 mg/kg). Among fruit examined quercetin concentrationaverages 15 mg/kg, with apples having the highest concentration of 21 to72 mg/kg (Hertog et al, 1992 J. Ag. Food Chem. 40, 2379-83).

Flavonol glycosides are present in grapes and values ranging from 8 to97 mg/Kg fresh weight have been reported (Macheix et al, 1990 FruitPhenolics pp 378 CRC Press Boca Raton). During the fermentation process,some of the sugar is split off and the aglycone formed. On average about50% of the flavonol exists in wine as the aglycone. The flavonol contentof grape skins and wine is very variable and depends on the variety ofgrape and more especially the amount of sunshine in which the grapes aregrown. The flavonols have a yellow color and act as filters to blue andultraviolet light which is very injurious to the grape. During periodsof intense sunlight more flavonols are synthesized to protect the grape,and consequently the grape skins and the wine from which it is made hasa high concentration of flavonols (Price et al, Am. J. Enol. Vitic. 46187-194, 1995).

Some wines in France have only 5 mg/L flavonol (calculated as aglycone)whereas up to 150 mg/L have been reported in some Californian wines. Theflavonols are virtually absent from the pulp and grape seeds and onlytrace quantities are present in commercial anthocyanin powders extractedfrom pomace after making red wine.

Although grape juice is often less rich in polyphenols than wine, itcontains flavonols and is a readily available commodity, and can also beused as a source of polyphenols.

Most flavonols enter the diet as glycosides with the glucosides andrutin as the most common flavonols consumed (Bokkenheuser and Winter,1988 Prog. Clin. Biol. Res., 280 142-148). The absorption of flavonolsin man has been a matter of some controversy. It was estimated that theabsorption of quercetin aglycone was only 0.3 to 0.5%. Thus with anintake of 50 mg, only some 0.25 mg would be absorbed (Formica et al Fd.Chem. Toxic. 1995 33 1061-1080). However recent work in ileostomysubjects showed that the absorption of quercetin aglycone was 25% andthe absorption of quercetin from onions (where it exists chiefly as theglucoside) was 52% (Hollman et al Am. J. Clin. Nutr. 1995 62,1276-1282). More recently it has been shown that the glucoside is morerapidly absorbed than the rutinoside (Hollman et al, 1997 Crit. Reviewsin Science & Nutrition 37, 719-738). One of the reasons for the poorabsorption of flavonols either as the aglycone or the glycosides is thatthey are almost insoluble in water and lipid solvents, althoughsparingly soluble in ethanol. Thus, on oral administration, a very highproportion of the flavonols are excreted because they are in a physicalstate which does not favour absorption. Thus, whilst the flavonols areconsidered pharmacologically beneficial, they are poorly absorbedbecause they are generally of low solubility. If the flavonols could bemade available in a water-soluble form they would be absorbed moreeasily and be biologically available following oral consumption.

The anthocyanins are water soluble pigments which are responsible forthe attractive colors of many flowers, fruit and leaves. They can beeasily extracted from plants by acidified alcoholic solvents and manyare available commercially as food colorants. They are usually suppliedwith malto dextrin as a diluent in a concentration suitable forinclusion in beverages or other foods (Timberlake 1980 Food Chemistry 5,69-80).

The anthocyanins are known to react with the flavonol glycosides in aphenomenon which is described as co-pigmentation (Scheffeldt et al 1978,J. Food Science 43, 517-520). For example, rutin was found to intensifythe colour of malvidin—3, 5, diglycoside and to shift the maximum peakof absorption. The process is believed to be the result of a hydrogenbonding mechanism.

Although the flavonols are insoluble in water, they exist in wine inquite high concentrations which vary according to the type of grape usedand country of origin (McDonald et al, J. Agric. Food Chem. 1998 46,368-375). The concentration of total flavonols (expressed as thealgycone) in wine was found to range from 4.6 to 41.6 mg/L. Since winecontains about 12% ethanol, and flavonols are soluble in ethanol, it wasreasonable to suppose that the relatively high solubility of flavonolsin wine was due to the presence of ethanol.

It would be a great advantage to provide flavonols for oraladministration in a soluble form, in water or aqueous mixtures oralternatively to increase the concentration of flavonols in ethanolcontaining beverages. Being in a soluble form would make possible theuse of flavonols in beverages, and potentially increase theirbio-availability.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a method of increasing thesolubility in water of a flavonol component of a flavonol-containingcomposition, the method comprising the steps of: providing aflavonol-containing composition; providing an anthocyanin-containingcomposition; and mixing the two compositions. The inventor has foundthat the presence of anthocyanin in a composition significantlyincreases the water-solubility (and hence bio-availability) offlavonols, especially at neutral or acidic pH values.

In a second aspect, the invention provides a method of providing anaqueous solution comprising dissolved flavonols at a concentration inexcess of 10 mg/L (preferably over 20 mg/L, more preferably over 50mg/L, and most preferably over 100 mg/L), the method including the stepsof mixing, in any order, water, a flavonol and an anthocyanin; andforming an aqueous solution from the mixture comprising the flavonoldissolved in the solution at a concentration in excess of 10 mg/L.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described by way of illustrative example andwith reference to the accompanying drawings, in which:

FIG. 1 is a schematic illustration of the core structure of flavones;

FIG. 2 is a schematic illustration of the core structure of flavanols;

FIG. 3 is a schematic illustration of the core structure ofanthocyanins;

FIG. 4 shows a graph of the solubility of quercetin in an aqueoussolution of grape-derived anthocyanin powder (containing 3.94%anthocyanin), following the addition of excess quercetin in an ethanolicsolution;

FIG. 5 shows a graph of the solubility of quercetin in an aqueoussolution of grape-derived anthocyanin powder (containing 3.94%anthocyanin) in which excess solid quercetin is heated with saidsolution;

FIG. 6 shows a graph of the solubility of quercetin in an aqueoussolution of black carrot-derived anthocyanin powder (containing 1.21%anthocyanin) in which excess solid quercetin is heated with saidsolution;

FIG. 7 shows a graph of the solubility of quercetin in an aqueoussolution of red cabbage-derived anthocyanin powder (containing 2.51%anthocyanin) in which excess solid quercetin is heated with saidsolution; and

FIG. 8 shows a graph of the solubility of quercetin in an aqueoussolution of malvin chloride, following the addition of quercetin in anethanolic solution.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the invention provides a method of increasing thesolubility in water of a flavonol component of a flavonol-containingcomposition, the method comprising the steps of: providing aflavonol-containing composition; providing an anthocyanin-containingcomposition; and mixing the two compositions. The inventor has foundthat the presence of anthocyanin in a composition significantlyincreases the water-solubility (and hence bio-availability) offlavonols, especially at neutral or acidic pH values.

The term ‘flavonol’ as used herein refers to compounds having thestructure shown in FIG. 1 and being hydroxylated at one or more ofpositions 3, 5, 7, 3′ 4′ or 5′. The term ‘flavonol’ is also intended toencompass associated derivatives which are additionally substituted,especially those compounds which comprise one or more sugar residues(such as glucose, galactose, arabinose, rhamnose and the like),particularly O-glycosylated compounds. Other compounds, falling withinthe definition of “flavonol” as used herein, include those comprisingother substituent groups such as alkyl (especially methyl), alkoxy(especially methoxy) sulphyl and malonyl groups.

Preferably the flavonol content of the composition comprises thecompound kaempferol, myricetin or, most preferably, quercetin or astructural analogue thereof. It will be understood from the foregoingthat reference to quercetin is intended to encompass the aglycone, orany glycoside thereof (typically an O-linked glycoside). The glycosideof quercetin tend to have acquired their own trivial names. For example,the rhamnose glycoside of quercetin is known as quercitrin, and therutinoside is known as rutin. Analogues of quercetin include thosecompounds which comprise a substituting group other than an —OH group atone or more of the positions 3, 5, 7, 3′ and 4′. Other possiblesubstituting groups include: lower alkyl (i.e. less than 5 carbon atoms)especially methyl or ethyl; acetyl; sulphyl; and malonyl. Desirably, inanalogues of quercetin only a few (i.e. preferably only one or two) ofthe positions are substituted with anything other than —OH groups.

Flavonols such as quercetin and glycosides thereof, such as quercetinglucosides, quercitrin (quercetin rhamnoside) and rutin (quercetinrutinoside), are readily available from chemical suppliers (e.g. Sigma,UK and Indofine Inc, USA) ranging from 85-99% purity.

[The glycosides can be expressed as the aglycone by multiplying theirconcentration by:$\left( {{{FW}\quad {{quercetin} \div {FW}}\quad {quercitrin}} = {\frac{302}{448} = 0.674}} \right)$

(FW=formula weight). The total flavonol content is the sum of theaglycone contents in the free and conjugated forms.]

Alternatively, as flavonols in general (including quercetin) are presentin many naturally occurring substances, such as foodstuffs, thecomposition of the invention may comprise a flavonol (especiallyquercetin) obtained from a naturally occurring source. Foodstuffs whichcontain quercetin in relatively large amounts include certain fruit andvegetables, such as apples, onions and red wine. In particulartherefore, the composition of the invention may comprise a flavonolcontent derived from plants. By way of explanation, such plant-derivedcompositions may comprise extracts of plants or parts thereof (such astubers, fruit) which are processed in some way (e.g. by fermentation).Thus, plant-derived compositions include aqueous or organic solventextracts of plants or parts thereof, fruit juices and fermented liquors(e.g. wine) produced from plants or fruit juice, or compositionsobtained from any of the foregoing. The plant material is typicallyprocessed (physically and/or chemically) during production of thecomposition to extract flavonols from the plant and so increase andenrich the flavonol content of the composition. It will be appreciatedby those skilled in the art that the invention is not intended toencompass within its scope naturally occurring foodstuffs or plantmaterials which have not been subjected to processing in any way.

Advantageously the flavonol-containing composition may be such that theplant-derived material comprises at least 25% polyphenols, morepreferably 35% polyphenols or most preferably at least 45% polyphenols.

The flavonol-containing composition may be comprised wholly orsubstantially of the plant-derived material from which the flavonolcontent of the composition is obtained. Alternatively, the compositionmay comprise other material, such as flavorings, excipients, carriersand the like conventionally used in formulating compositions for humanconsumption.

Typically the flavonol content of the composition as a whole is at least0.1% w/w, preferably at least 1% w/w, more preferably at least 5% w/w,and most preferably at least 10% w/w.

The term “anthocyanin” as used herein is intended to refer not only tomonomeric anthocyanins, but also refers to dimeric and polymeric (i.e.containing from 3 to 20 anthocyanidin monomer residues) forms ofanthocyanins and to leucoanthocyanidins (also known as flavan-3,4-diols). The anthocyanins may comprise substitutions (e.g. alkyl,alkoxy groups etc.) and in particular may be O-glycosylated, in a mannersimilar to that of the flavonols, as described above.

The amount of anthocyanin added to the composition should ideally becarefully controlled. As anthocyanins are generally highly pigmented,their presence in high concentrations in certain foods, drinks or foodsupplements may be undesirable if it is preferred to avoid the colorassociated with the presence of anthocyanins. Accordingly it may bepreferred in many embodiments to add the minimum amount of anthocyaninconsistent with achieving the required level of solubilisation offlavonol. Additionally, or alternatively, it may be desirable to includesome other component which masks or hides the color associated with theanthocyanin—such a component could conveniently be another coloring orother substance suitable for human consumption.

The anthocyanin in the composition may be a single anthocyanin, orcomprise a mixture of anthocyanins. Preferably the anthocyanin isselected from the group consisting of: malvidin, cyanidin, delphinidin,paeonidin, pelargonidin and petunidin, and glycosides thereof A typicalexample is malvin (malvidin diglucoside) chloride, which is commerciallyavailable in a purified form. Alternatively the anthocyanin may beobtained by extracting anthocyanin containing plants such as grape,black carrot, red cabbage, blackberry, blackcurrent, cranberry and thelike.

Those skilled in the art will know of the many methods which areemployed for extracting anthocyanins since they are widely used forpreparing food colorants. The anthocyanin in the composition can be inhighly purified form, or a mixture with other polyphenols especiallyflavonols, or with a diluent such as maltodextrin. It is preferred thatthe content of anthocyanin in the composition as a whole is at least 1%(w/w), more preferably at least 5%, and most preferably at least 10%.The content of anthocyanin in the composition should typically besufficient to solubilize in water at least 0.1% w/w flavonol, preferablysufficient to solubilise at least 1% w/w flavonol in said composition.

Preferably, Where the method involves mixing of a flavonol-containingcomposition with an aqueous solution of anthocyanins, the aqueoussolution will conveniently comprise anthocyanin dissolved at aconcentration of at least 10 mg/L, more preferably at least 50 mg/L, andmost preferably at least 100 mg/L.

It will be apparent to those skilled in the art that the order ofaddition of the flavonol and anthocyanins is not critical. Thus, forexample, one may prepare an anthocyanin-containing composition and addto that composition a suitable amount of flavonol, or vice versa. Ingeneral however, in view of the poor solubility of flavonols, it willnormally be preferred to add the flavonol-containing composition to theanthocyanin-containing composition such that the anthocyanins will bepresent in excess, at least initially, which will tend to preventclumping of solid flavonol in the mixture and so facilitate mixing.

It may be preferred that a slight excess of anthocyanin will be presentafter mixing of the two compositions has been completed, so as to ensuresolubilisation of at least the desired amount of flavonol.

In one particular embodiment, the method comprises the fortification ofcompositions (such as red wine or dealcoholised red wine) withadditional flavonol, simply by the addition of extra flavonol to apre-existing flavonol/anthocyanin composition (e.g. red wine), providedthat there is sufficient anthocyanin in the pre-existing composition tosolubilise the extra added flavonol. If there is insufficient excessanthocyanin already in the pre-existing composition, one may simply adda suitable additional amount of additional anthocyanin.

However wines are comparatively expensive materials, and anthocyaninsare present in high concentrations in fruit and vegetable juices, suchas grape, cranberry, blackcurrant and blackberry juices. In preferredembodiments the invention will comprise the addition of flavonols (suchas quercetin) to fruit and/or vegetable juices, so as to fortify thefruit or vegetable juice with additional flavonol.

In some embodiments it may be desirable to facilitate initialdissolution of the flavonols. This can be achieved, for example, byadding ethanol to the composition or by initially dissolving theflavonols in a small amount of ethanol and adding the ethanol-dissolvedflavonols to the rest of the composition. Additionally or alternatively,the composition may be heated (e.g. to 50° C. for about 30 minutes, orto 75° C. for about 10 minutes, or to 100° C. for about 1 minute). Theapplication of heat may be especially preferred if it is desired toprovide an ethanol-free flavonol containing composition.

The composition prepared by the method of the invention, when dry, istypically solid at atmospheric pressure (760 mm Hg) throughout thetemperature range 10-20° C. The composition may be particulate (e.g.powdered or granulated), or may be formed into capsules, tablets and thelike.

Alternatively the composition may be a liquid comprising water or otheraqueous solvent such as aqueous ethanol. The inventor has surprisinglyfound that compositions prepared in accordance with the method of theinvention present flavonols in a more soluble form in aqueous solutions.Flavonols are almost insoluble in water at pH 7.0 or below, but in thepresence of anthocyanin, their solubility is markedly increased, and theflavonols are rendered bio-available.

Moreover, when a composition containing both a flavonol (e.g. quercetin)and anthocyanin is administered to volunteers for 4 weeks, the plasmaantioxidant capacity of the volunteers is significantly increased (asdetermined by the FRAP assay, described below).

The flavonol-containing composition may comprise flavonols (includingquercetin) obtained from grapes (whole grapes or parts thereof, such asskins or juice), wine (especially red wine, which comprises much higherconcentrations of polyphenols than white wine), or by-products and/orwaste products of the wine-making process, such as pomace (i.e. theresidue of crushed grapes following juice extraction) or marc (wastesolids remaining after initial fermentation). However, flavonols such asquercetin are present in a wide range of naturally occurring materials,many of which contain a higher flavonol content than red wine and areconsiderably cheaper, and so present more appropriate sources offlavonol. Examples of such materials include: fruit in general, such asapples (e.g. var. “Gravensteiner”); especially apple peel; pears (e.g.var. “Williams Christs”); bell peppers (e.g. var. “Yolo wonder”;redcurrants; blackcurrants (particularly preferred as being relativelyhigh in flavonols); lemons; cherries; cranberries; gooseberries;tomatoes; olives; and vegetables in general, including: radishes (e.g.var. “Saxa treib”); kohlrabi (e.vg. var. “Primavera”); horseradish;potatoes; onions; and asparagus.

In a one embodiment of the invention, the flavonol-containingcomposition is derived from a red wine and comprises a representativeprofile of substantially all the flavonol compounds present in the wine(typically, although not necessarily, present in the compositionsubstantially in the relative amounts representative of those in thewine from which the composition is derived). Such a composition may bereferred to as a “total flavonol pool” of the wine.

Flavonols may conveniently be obtained from red wine or otherflavonol-containing liquids (such as fruit or vegetable extracts) byabsorption onto a chromatographic resin column, with elution of thepolyphenol-enriched fraction from the column (typically following awashing step) by use of a 40-50% ethanol eluent, or other suitableorganic solvent (such as methanol, acetone, ethyl acetate, dimethylenechloride, and chloroform—which may be in aqueous solution). The organicsolvent is preferably relatively volatile (i.e. having a boiling pointof between 30 and 85° C. at 760 mm Hg pressure) and so readily drivenoff, to leave a substantially dry (i.e. less than 10% w/w H₂O) solidcomposition comprising flavonols. Such a method may successfully be usedto obtain a total flavonol pool from red wine.

Alternatively, flavonols may be obtained from red wine or otherflavonol-containing liquid by solvent extraction using a suitableorganic solvent immiscible with the wine or other liquid. Alternatively,flavonols may be obtained from flavonol-containing solids by solventextraction (typically extraction with an organic solvent such as ethanolor ethyl acetate)—the solid can then be separated from the solvent byfiltration or centrifugation. The solvent may then be evaporated toleave a substantially dry, solid composition comprising flavonols.

Alternatively the flavonols may be obtained in purified form (e.g.85-99% pure) from a chemical manufacturer, providing that the materialis suitable for human consumption.

In preferred embodiments, the composition obtained by mixing theflavonol-containing and the anthocyanin-containing compositions ispresented as a food supplement. This may be a substance to add as anadditional ingredient during manufacture of the foodstuff, or may be aseparate substance to be consumed by an individual (e.g. as a tablet orcapsule) substantially in isolation from (i.e. not mixed with) otherfood components prior to consumption (although, of course, the tablet orcapsule may be taken with food). The invention thus includes within itsscope a prepared product, particularly a prepared foodstuff (i.e. onewhich is not naturally occurring) comprising a composition in accordancewith the invention, which may be in the form of a solid or a drink (e.g.a flavonol-fortified fruit or vegetable juice). Alternatively, thecomposition may be presented as a solid to be made into a drink bymixing with a physiologically acceptable diluent (such as milk, water,solutions or gels prepared from Aloe vera, or other aqueous liquid).

The dosage of composition given to a subject is dependent on the degreeof activity of the material but will normally be between 10 mg and 10 gper day. The preferred dose of flavonol will be in the range 0.1-1000 mgper day, preferably in the range 0.5-500 mg per day, more preferably inthe range 2-250 mg per day.

Compositions obtained by mixing the flavonol-containing andanthocyanin-containing compositions in accordance with the invention,may be prepared in accordance with conventional food supplement orpharmaceutical practice. The diluents, excipients or carriers etc. whichmay be used are well known in the formulation art and the form chosenfor any particular regimen will depend on the given context and theformulator's preferences. In general the dose will depend on theconcentration of flavonols (particularly quercetin) in the composition,and the identity of the flavonol compounds in question.

Moreover, the compositions may comprise any number of furthercomponents, such as those typically used in the food industry and/or inthe pharmaceutical industry. Such components may include nutrients(especially trace elements and vitamins), antioxidants, therapeuticsubstances (especially those having a therapeutic effect in relation toprevention and/or treatment of CHD, in particular, aspirin), flavoring,and sweeteners (especially artificial sweeteners, such as aspartameetc.).

Examples of the above include the following: a carotenoid such aslutein, lycopene, or α-and/or β-carotene; antioxidant nutrients oranti-inflammatory agents such as vitamin A, vitamin C, vitaminE(α-tocopherol and other active tocopherols), folic acid, selenium,copper, zinc, manganese, ubiquinone (coenzyme Q10), salicylic acid,2,3-dihydroxy benzoic acid, and 2,5-dihydroxy benzoic acid.

Antioxidants such as carotenoids and vitamin E are partially destroyedin the gastro-intestinal tract by oxidation. By inclusion of thesecompounds in the composition of the invention it is believed that thisprocess is inhibited and more antioxidants are absorbed. Use of acomposition comprising α-tocopherol and/or aspirin is especiallypreferred since it is believed that such a mixture afforts asynergistic-effect in the presence of flavonols.

Typical suitable daily dose of these additional components of thecomposition (and which may therefore be included in the composition suchthat normal consumption of the composition will give the appropriatedose) are as follows:

Lutein 2 to 50 mg e.g. conveniently 7.5 mg Beta carotene 2 to 20 mg e.g.conveniently 5 mg Vitamin A 400 to 600 RE e.g. conveniently 500 REVitamin C 75 to 250 mg e.g. conveniently 100 mg Folic Acid 0.1 to 1.0 mge.g. conveniently 0.2 mg Selenium 80 to 120 μg e.g. conveniently 90 μgCopper 2 to 4 mg e.g. conveniently 3 mg Zinc 10 to 20 mg e.g.conveniently 15 mg Coenzyme Q10 10 to 200 mg e.g. conveniently 30 mgAspirin 10 to 150 mg e.g. conveniently 75 mg

Thus, in one embodiment the composition takes the form of capsules, eachcapsule containing 50 mg of flavonol composition, with a suggestedintake of one to four capsules per day. Another presentation is as anon-alcoholic drink which provides an effective dose of flavonols whendissolved in water (still or aerated) flavored and sweetened to taste,or dissolved in a fruit juice e.g. grape, apple or orange etc., or insolutions or gels prepared from Aloe vera.

Whilst it may be preferred for a number of reasons (e.g. social,religious and economic) to provide an alcohol-free drink comprising thecomposition of the invention, such drinks can be fortified with alcohol(e.g. from vodka, gin, whisky) to give a desirable level of 5-50%alcohol depending on the consumer's taste.

Other presentations are as a food ingredient in dairy products such asmilk and yogurts, preserves, and dietary products intended as mealsupplements or replacements. The above examples are illustrative onlyand are not intended to be limiting in any way.

In a second aspect, the invention provides a method of providing anaqueous solution comprising dissolved flavonols at a concentration inexcess of 10 mg/L (preferably over 20 mg/L, more preferably over 50mg/L, and most preferably over 100 mg/L), the method including the stepsof mixing, in any order, water, a flavonol and an anthocyanin; andforming an aqueous solution from the mixture comprising the flavonoldissolved in the solution at a concentration in excess of 10 mg/L.

Conveniently the aqueous solution does not contain any organic solventand in particular is preferably substantially free of ethanol, such thatthe solubilisation of the flavonol in water is primarily due to thepresence of the anthocyanin.

The aqueous solution prepared by the method of the second aspect of theinvention is safe for human consumption, and will typically beformulated as a drink, comprising optional ingredients such asanti-oxidants, flavoring agents, sweeteners, vitamins or minerals asdescribed previously.

As explained previously, heat may advantageously be used to facilitatethe initial dissolution of the flavonols, which substantially remain insolution upon cooling to room temperature. Alternatively, and lesspreferably, ethanol may be used to assist dissolving the flavonols. Themethod may be particularly advantageous when producing an aqueoussolution of dissolved flavonols at neutral or acidic pH.

EXAMPLES Example 1 The Solubilization of Quercetin in Wine and FruitJuices by the Addition of an Ethanolic Solution of Quercetin

The purpose of the investigation was to determine the solubility ofquercetin in red wine, a white wine and two fruit juices when the addedquercetin was dissolved in ethanol. The quercitin used in this exampleand in others was quercetin dihydrate (C₁₅H₁₄O₉, FW 238, Sigma, UK).

The solubility of quercetin is expressed as the anhydrous molecule(C₁₅H₁₀O₇, FW 302). An ethanolic solution of quercetin was prepared andadded in slight excess to red wine, white wine, cranberry juice andblackcurrant juice, at room temperature. After centrifugation to removesolid quercitin, the optical density was measured in a spectrophotometerat 258 nm and 375 nm. At these wavelengths quercetin has maximumabsorption and its concentration can be calculated using the factorlog_(epsilon)=2.75.

The procedure was as follows: a stock solution of quercetin dihydrate inabsolute ethanol was prepared containing 5 mg quercetin dihydrate perml. Aliquots of 50 μL of this solution were added to 5ml of the liquidsbeing examined. The mixtures were then vigorously stirred on a Vortexmixer and allowed to stand for 20 to 30 min at room temperature for aprecipitate to form.

The mixture was then centrifuged at 2000×g for 30 min at 20° C. Aliquotswere then taken and the optical density measured in a spectrophotometer(Camlab, UK) at 258 nm and 375 nm, diluting where necessary with anappropriate diluent e.g. water for fruit juices, 12% ethanol for winesetc. In addition, 50 μL ethanol was added to 5 ml of each of the liquidsbeing examined and the optical densities determined likewise. Thecontrols were water and 12% ethanol. The solubilities of quercetin inthe liquids were determined from the differences between the opticaldensities of the liquids with and without quercetin.

The solubility of quercetin in the wines and fruit juices examined areshown in Table 1.

Quercetin is almost insoluble in water (1 mg/L) and very sparinglysoluble in 12% ethanol (5 mg/L). However quercetin is much more solublein red wine (40 mg/L), cranberry (35 mg/L) and blackcurrant juices (28mg/L). Quercetin was not more soluble in white wine (5 mg/L) than in 12%ethanol.

TABLE 1 Solubility of quercetin in fruit juices, after the addition ofan ethanolic solution of quercetin Solubility of Quercetin^(a) mg/LLiquid 258 nm^(b) 375 nm^(b) Red wine^(c) (Cabernet Sauvignon) 44.1 39.8White wine^(c) (Vin Ordinaire) 4.2 5.4 Cranberry juice^(d) 33.6 34.7Blackcurrant juice^(e) 28.1 31.0 Ethanol 12% v/v 4.1 5.3 Water 2.2 1.8^(a)Values expressed as the anhydrous compound ^(b)Wavelengths ofoptical density measurement ^(c)French wines ^(d)Ocean Spray ™ brand^(e)Ribena ™ brand

Since white wine does not contain significant amounts of polyphenols, itwould appear that the quercitin is solubilized by polyphenols present inred wine and fruit juices, possibly by formation of a soluble complex.

The method of adding quercitin to a red wine or fruit juices containingpolyphenols is a practical means of fortifying drinks with a flavonol,such that the flavonol is available in solution. Because flavonols arealmost insoluble in water or dilute aqueous ethanol it is impracticableto provide flavonols in these solvents since if solid flavonol is addedmost of it does not dissolve but sinks to the bottom of the vessel.

Example 2 The Solubilization of Quercetin in an Aqueous Solution ofGrape Anthocyanin, following the Addition of Excess Quercetin in anEthanolic Solution

The purpose of this investigation was to examine the solubility ofquercitin in grape anthocyanin which is a major component of thepolyphenols in red wine and coloured fruit juices.

The grape anthocyanin powder was a commercial product used as a foodcolorant (Phytone Ltd, Burton on Trent, UK) and had an anthocyanincontent of 3.94%.

The procedure used was as follows: anthocyanin powder was dissolved indeionised water to give concentrations of 0.625, 1.25, 2.5, 10 and 20mg/ml. A stock solution of quercetin dihydrate (5 mg/ml) in absoluteethanol was also prepared. To each 5 ml of the above anthocyaninsolution was added 133 μL of the quercetin solution at room temperature,followed by increasing aliquots of 30 μL until a precipitate was clearlyvisible. The mixtures were allowed to stand for 20 to 30 min and thencentrifuged at 2000×g for 30 min at 20° C. The optical densities weremeasured at 258 nm and 375 nm as in Example 1. To 5 ml of eachcorresponding anthocyanin solution was added the same volume of ethanoldetermined above to give a precipitate, and the optical densitiesmeasured. The concentration of quercetin was determined from thedifference in optical density between the solutions with or withoutquercetin. The control was water.

Since quercitin is more soluble in aqueous ethanol than water a furtherseries of controls were prepared in which quercetin in ethanol was addedto water in amounts corresponding to those used in the test, and thesolubilities likewise determined. As shown in FIG. 4 the solubility ofquercetin increased in almost linear fashion with increasingconcentration of anthocyanin powder (at least at low [<10 mg/ml]anthocyanin powder concentration). With 20 mg anthocyanin powder/ml thesolubility of quercetin was 75 mg/L. This was in a 22% ethanol solution.Without anthocyanin present the solubility of quercetin is only 13 mg/L.The commercial anthocyanin powder contained 3.94% anthocyanin. Thus inthe 20 mg/ml solution the concentration of anthocyanin was 800 mg/L.Thus the ratio of quercetin: anthocyanin in the solution was 1:10.7. Tosolubilize quercetin more anthocyanin was needed than quercetin w/w.

It is concluded that flavonols are readily soluble in aqueous solutionof anthocyanin when the quercetin is added as an ethanolic solution.Since anthocyanin powders are readily available commercially, it ispractical to supplement drinks containing anthocyanin with quercetin ina soluble form.

Example 3 The solubilization of Quercetin in Different AnthocyaninContaining Liquids by Means of Heat

Although it is convenient to add quercetin in ethanolic solution toanthocyanins, the method increased the ethanol content of the beverage.An investigation was made into the possibility of dissolving quercetin,without the use of ethanol.

The procedure was follows: aliquots (20 ml) of the liquids examined wereplaced in a plastic tube with 50 mg solid quercetin dihydrate androtated at room temperature (20° C.) for 3 hours followed bycentrifugation at 2000×g for 30 min at 20° C. Quercetin concentrationwas measured as in previous examples from the difference in opticaldensity at 375 nm of liquids with and without quercitin.

The solubility of quercetin when mixed with anthocyanin containingsolutions at room temperature was extremely small (Table 2). For redwine the solubility of quercetin was only 3 mg/L and for grapeanthocyanin powder (1 g/L) only 0.7 mg/L.

To investigate dissolution by heat, the liquids were heated in a sealedtube for 1 min in a microwave oven (when boiling of the liquid occurred)and cooled to room temperature. Under these conditions (see Table 2),quercetin was much more soluble, and the solubility of quercetin wascomparable to that achieved by the previous methods using ethanolicsolution of quercitin (Table 1).

Anthocyanin concentrations of the liquids were also determined by themethod of Miketic-Aleksic et al 1972, Lebensm-Wiss.u Technol. 5163-165). The method consists of diluting 1 ml aliquots of wine or juicewith a buffer at pH1 and measuring the optical density at 520 nm.Results were calculated as malvidin-3-glucoside (FW. 493.5) using theextinction coefficient of 28,000. For solids, a known weight of powderwas dissolved in buffer at pH1 and the results obtained as ganthocyanin/100 g powder.

Samples of red wine, grape anthocyanin and grape seed extract wereanalyzed for polyphenols by means of HPLC by ETS Laboratories, StHelena, Calif. 94574, USA. The four red wines and rose wine all produceda higher solubility of quercetin than in 12% ethanol. The three winesabove 150 g anthocyanin/L gave a greater solubility than the two winesbelow 150 g anthocyanin/L. There was no increase in the solubility ofquercetin in white wine (in which anthocyanin is substantially absent).The highest solubility was in blackcurrant juice, which also had thehighest anthocyanin content. The four fruit juices all containedanthocyanin and gave an increase in solubility of quercetin. Quercitinalso demonstrated increased solubility in a grape anthocyanin solution,which was comparable to that of the wines and fruit juices. White winesin which anthocyanin was absent were similar in effect to 12% ethanol.

Whilst anthocyanin powder had a positive effect on solubility, grapeseed extract which does not contain anthocyanin was ineffective.

The method of providing a beverage enriched in flavonol is simple andrequires only a brief period of heating. Moreover it leaves the liquidsrelatively unchanged in composition, and does not increase the ethanolcontent.

Table 3 shows results of the polyphenol analysis by HPLC. All threesamples, red wine, grape anthocyanin and grape seed extract, containedappreciable amounts of polymeric phenols. Red wine and grape seedextract contained flavanols (catechin, epicatechin) and procyanidindimers. Red wine and grape anthocyanin contained anthocyanin monomersand polymers. Since grape seed extract was inactive in increasing thesolubility of quercetin, it was concluded that it is the anthocyaninswhich are the solubilizing factor, and these are present in red wine andthe grape anthocyanin powder, but are absent from the grape seedextract.

TABLE 2 Solubility of quercetin in different liquids Solubility ofquercetin Anthocyanin mg/L present Liquids Room temp Boiling mg/LControls Water 0.3  2  0 Ethanol 12% v/v 1  3  0 Red wine^(b)Cabernet-Sauvignon (F) 3 36 196 Cabernet-Sauvignon (ARG) 18 224 Rhone(F) 19 271 Shiraz (AUS) 11 118 Rosé^(b) Rosé D'Anjou (F)  9 138White^(b) Vin Ordinaire (F) 0  4  0 Chardonnay (AUS)  6  0 Fruit juicesCranberry^(c) 0 12 109 Cranberry-blackcurrant^(c) 16 155 Grape juice^(d)38 152 Blackcurrant^(e) 82 307 Polyphenol extracts in waterAnthocyanin^(f) 1 g/L 0.7 50 394 Grape seed^(g) 125 g/L  4  0 250 g/L  4 0 500 g/L  6  0 ^(a)measured from O.D. at 375 nm ^(b)origin of wines: F= France, ARG = Argentine AUS = Australia ^(c)Ocean spray ™ brand^(d)Welch's ™ Concord brand ^(e)Ribena ™ ^(f)Vinox ™ (Polyphenolics Inc)solid dissolved in water ^(g)Phytone Ltd solid dissolved in water

TABLE 3 Polyphenols in red wine, anthocyanin powder and grape seedextract Grape anthocyanin Grape Red wine powder seed extract Polyphenolsmg/L mg/g mg/g 1 gallic acid 35 0.9 3.9 2 procyanidin dimers 49 <0.177.0 3 catechin 20 <0.1 74.6 4 epicatechin 15 <0.1 51.0 5 syringic acid 8 <0.1 <0.1 6 polymeric phenols 445  50.0 356 7 caftaric acid 12 0.2<0.1 8 caffeic acid  5 <0.1 <0.1 9 coutaric acid 14 <0.1 <0.1 10p-coumaric acid  5 <0.1 <0.1 11 trans resveratrol  9 <0.1 <0.1 12 cisresveratrol  2 <0.1 <0.1 13 myricetin glycosides  2 <0.1 <0.1 14quercetin glycosides  8 0.5 1.8 15 myricetin  1 <0.1 <0.1 16 quercitin 1 <0.1 0.2 17 delphinidin glycoside  2 4.4 <0.1 18 cyanidin glycoside<1 1.7 <0.1 19 peonidin glycoside  2 5.2 <0.1 20 petunidin glycoside  22.8 <0.1 21 malvidin glycoside  7 14.0 <0.1 22 polymeric anthocyanins 224.4 <0.1

Example 4 The Solubilization of Quercetin by Heating Excess QuercetinSolid With Aqueous Solutions of Three Different Commercial Preparationsof Anthocyanins Obtained from Grape, Blackcurrant and Red Cabbage.

The purpose of the investigation was to compare the ability ofanthocyanin from different sources to solubilize quercitin.

The procedure was as follows: The grape anthocyanin was obtained fromPhytone Ltd, UK (as used in example 2). Blackcurrant and red cabbageanthocyanin were obtained from Overseal Ltd, Swadlincote Derbyshire UK.The anthocyanin contents of each powder were measured according to themethod of Niketic-Aleksic et al (as in example 3).

Solutions of the anthocyanins were made in water ranging from 2.5 to 30mg/ml for grape, 15-40 mg/ml for black carrot and 25 to 150 mg/ml forred cabbage. To 20 ml of each solution was added 50 mg quercetindihydrate solid and the mixture heated for 1 min in a microwave oven asbefore, cooled to room temperature and centrifuged at 2000×g for 30 min.Aliquots with and without quercetin were diluted with water 1:9 and theoptical density read at 375 nm. The concentration of quercetin wasmeasured by difference in optical density between solutions with andwithout quercetin as previously. As shown in FIGS. 5 to 7 quercetin wasmore soluble in all three solutions of anthocyanins than in water aloneand the effect was substantially linear with increasing concentrationsof anthocyanin powder. The anthocyanin content of the three powders weregrape: 3.94 g/100 g, black carrot 1.21 g/100 g, red cabbage 2.51 g/100g.

The results demonstrate that the increased solubility of quercetin inanthocyanins is a general phenomenon and not restricted only to grapeanthocyanins but can be obtained with other suitable edible materialthat contains anthocyanins.

Example 5 The Solubilization of Quercetin in an Aqueous Solution ofMalvin Chloride by the Addition of an Ethanol Solution of Quercitin.

The anthocyanins can be divided into two major classes: the monomericanthocyanins and the polymeric anthocyanins. A typical example of amonomeric anthocyanin is malvin chloride, a salt of the oxonium ionmalvidin-3-glucose and the chloride ion. It can be obtained in a highstate of purity (Indofine Inc, USA) and therefore used to determine thesolubilizing effect of an anthocyanin monomer. The procedure was asfollows: malvin chloride (2.2 mg) was dissolved in 2 ml citrate buffer(pH 3.0)—one ml acted as a control. To the other ml was added stepwise0.25 μl ethanol containing 5 mg/ml quercetin dihydrate. Equal amounts ofethanol were added to the control. Each solution was diluted (1:9) with5% aqueous ethanol and the optical density determined at 275 nm. Thequercetin concentration was calculated as in previous examples.

As shown in FIG. 8, up to 1000 μg quercetin was added to 1 ml malvinchloride. With 750 μg quercetin no precipitate of quercetin appeared,but there was a slight precipitate at 1000 μg which was centrifuged off.The concentration of quercetin in malvin chloride was linear up to 750μg.

At this concentration of added ethanol (20% v/v) quercetin is soluble tothe extent of 13 mg/L. Thus under these conditions, quercetin is madeextremely soluble by the presence of malvin chloride, although it isquite possible that the solution was super saturated since on standingat 4° C. a large precipitate occurred.

Example 6 A Comparison of the Solubilization of Quercetin, Quercitrinand Rutin by Heat in Solutions of Grape Anthocyanin and Malvin Chloride.

Quercetin is a flavonol aglycone. In fruit juices it exists as theglycoside. The purpose of the investigation was to examine thesolubilization of glycosides of flavonols by anthocyanins. The threeflavonols studied were quercetin dihydrate, quercitrin (quercetin3-L-rhamnoside, C₂₁, H₂₀, O₁₁, FW 448.4 Sigma UK) and rutin (quercetin3β-D-rutinoside hydrate, C₂₇, H₃₀, O₁₆, FW 610.5, Sigma, UK) Theprocedure was as follows: To 1 ml grape anthocyanin (20 mg/ml) in waterwas added 2.5 mg of the flavonol and the mixture heated for 1 min in amicrowave oven, cooled, centrifuged and the optical density of thesupernatant measured at 375 nm. Samples were diluted in water 1:9 beforereading. Aliquots of anthocyanin and water with and without the flavonolwere treated similarly.

Flavonol concentrations expressed as mg/L quercetin aglycone werecalculated from the differences between the solution with and withoutthe added flavonol, as previously.

The same procedure was used for measuring the solubility of theflavonols in malvin chloride. In this case 1.8 mg malvin chloride wasdissolved in 2.0 ml citrate buffer at pH3. To 1 ml was added 2 mg of theflavonol, the other 1 ml acted as a control. After heating, the mixturewas cooled to room temperature, centrifuged, and the supernatant diluted1:9 with 50% aqueous ethanol. The optical densities were measured at 375nm.

As shown in table 4, the solubility of quercitrin and rutin in water wasvery much higher than quercetin. The solubility of all the flavonolswere increased in the presence of grape anthocyanin and malvin chloride.This demonstrated that glycosides of quercetin were more soluble inanthocyanin solutions than in water. Moreover the increased solubilitywas also seen with a pure anthocyanin monomer, malvin chloride.

TABLE 4 A comparison of the solubilities of quercetin, quercitrin andrutin by heat in solutions of aqueous grape anthocyanin or malvinchloride. Grape Anthocyanin Malvin chloride Flavonol Water Solubility ofquercetin aglycone in mg/L Quercetin  2  90 28 Quercitrin 53 316 127 Rutin 36 260 74

Example 7 Preparation of Composition from a Red Wine Containing a LowConcentration of Flavonols and Enrichment with Pure Flavonol

The red wine processed was of the Cabernet Sauvignon variety fromFrance. It contained 1.8 g/L polyphenols. The flavonol content(calculated as the aglycone) was 8.8 mg/L (myricetin glycosides 1.1mg/L, quercetin glycosides 5.4 mg/L, myricetin 1 mg/L, quercetin 1mg/L).

The powder containing the polyphenols was made as follows: 1 litre ofred wine was filtered, rotary evaporated under reduced pressure at 75°C. for one minute, cooled, then rotary evaporated under reduced pressureat 55° C. until the volume was reduced to approximately 150 ml. 50 g ofDiaion™ resin (Supelco, UK) was weighed into a beaker covered withmethanol and allowed to stand for 15 minutes. The methanol was thendecanted off, and replaced with water. After standing for 10 minutes theresin was stacked into a glass column (half filling the column), andwashed with water.

The wine concentrate was applied to the column, followed by a water wash(300 ml). The polyphenols were eluted with 50% ethanol. Collection wasbegun as the colored fraction began to exit the column, and was endedwhen the eluate was free from color (after a total of 500 ml 50% ethanolhad been used). Phenolic content of the eluate at this point was at aconcentration of 0.1 mg/L as determined by the Folin-Ciocalteu method(Singleton & Rossi 1965 Amer. J. Enol. & Viticulture 16 144-158).

The phenolic fraction was reduced by rotary evaporation at 50° C. toapproximately 120 ml and this was freeze-dried to give the final sample.The yield of powder was 2.24 g/L with a polyphenol content of 45%. Theflavonol content was only 4.5 mg aglycone/g polyphenols (0.45% w/w).

Fortification of polyphenol powder with pure flavonol was made asfollows: 400 g of the polyphenol powder was dissolved in 10 L deionizedwater. Quercetin dihydrate (Sigma, UK) amounting to 12 g was dissolvedin 3.5 L ethanol and added to the above to give a clear solution. Thealcohol was removed by a rotary evaporator under vacuum, and the residuefreeze-dried to give a solid with 3-4% moisture content, which wasfreely soluble in water.

Example 8 The Effect of Adding Quercetin to a Preparation of Red WinePolyphenols and Administering said Mixture to Volunteers

The purpose of this investigation was to compare the effects of red winepolyphenols with and without added quercetin on the antioxidant capacityof plasma (FRAP) in volunteers.

13 healthy men aged 35 to 65 who were non-smokers, consuming a standardUK diet participated in a private and confidential study. Two weeksbefore the study the volunteers discontinued wine consumption. Allvolunteers were asked to maintain their usual diet and lifestyle duringthe study. The volunteers were divided into three groups A, B and C andgiven for 4 weeks one of the following—A: (a placebo drink consisting of36.6 g glucose, 1.1 g citric acid, 0.38 g blackcurrant flavor, 0.38 gtri-sodium citrate) in 300 ml water. B: A placebo drink as in Acontaining 1 g red wine polyphenols, prepared as in Example 7, or C: aplacebo drink as in A containing 1 g red wine polyphenol +30 mgquercetin (as prepared in Example 7). The trial commenced in the monthof November and ended in December.

Blood samples were drawn into K₃ EDTA (1 mmol/L) 12 h after their suppermeal before and after consuming the drinks for 4 weeks. Samples werecentrifuged at 2000×g for 15 min at 4° C. to obtain plasma.

Total plasma antioxidant capacity was determined by the Ferric ReducingAbility of Plasma (FRAP) assay (Benzie and Strain, 1996 AnalyticalBiochem 239, 70-76) on fresh plasma. The estimation was carried out onthe same day.

As shown in Table 5 there was a decrease of 10% in antioxidant capacitywith the placebo drink, and a decrease of 1% with the red winepolyphenol powder, and an increase of 6% with the polyphenol powder withadded quercetin.

It was concluded that supplementation of quercitin mixed with red winepolyphenols containing anthocyanin produced a greater increase inantioxidant capacity than the red wine polyphenols alone or a placebodrink. Moreover the beverage was fortified with a flavonol present in awater soluble form.

TABLE 5 Effect of administering a drink containing red wine polyphenolswith or without quercetin to volunteers on then antioxidative capacityof plasma (FRAP) FRAP^(a) B C A Red wine Red wine Placebo Polyphenolspolyphenols + quercetin Treatment (5)^(b) (4) (4) At start 1221 ± 2051277 ± 99 1151 ± 66 After 4 weeks 1148 ± 157 1260 ± 88 1215 ± 86Difference −127 17 64* ^(a)mean ± SD ^(b)no of subjects *P < 0.05 (A vs.C)

What I claim is:
 1. A method of increasing the solubility in water of aflavonol component of a flavonol-containing composition, the methodcomprising: providing a flavonol-containing composition; providing ananthocyanin-containing composition; and mixing the two compositions,said mixed composition having a greater concentration of anthocyaninover the concentration of flavonol.
 2. A method according to claim 1,wherein said mixed composition is a nutritional supplement.
 3. A methodaccording to claim 1, wherein said composition has a mean molar ratio ofanthocyanin to flavonol greater than 1:1.
 4. A method according to claim1, wherein the flavonol component comprises an aglycone or a glycoside.5. A method according to claim 1, wherein the flavonol componentcomprises a flavonol selected from the group consisting of quercetin,kaempferol, myricetin and glycosides thereof.
 6. A method according toclaim 1, wherein the anthocyanin-containing composition comprises afruit juice or vegetable extract.
 7. A method according to claim 1,wherein the anthocyanin-containing composition is obtained from thegroup consisting of grapes, cranberries, blackberries, andblackcurrants.
 8. A method according to claim 1, wherein theanthocyanin-containing composition is obtained from wine ordealcoholised wine.
 9. A method according to claim 1, wherein theanthocyanin is selected from the group consisting of malvidin, cyanidin,delphinidin, paconidin, pelargonidin, polymeric anthocyanins,leucocyanidins, and glycosides thereof.
 10. A method according to claim1, wherein a flavonol-containing composition is added to and dissolvedin an aqueous solution of anthocyanin.
 11. A method according to claim10, wherein the aqueous solution comprises dissolved anthocyanin at aconcentration of at least 10 mg/L.
 12. A method according to claim 10,wherein the aqueous solution comprises dissolved anthocyanin at aconcentration of at least 50 mg/L.
 13. A method according to claim 10,wherein the aqueous solution comprises dissolved anthocyanin at aconcentration of at least 100 mg/L.
 14. A method according to claim 10,wherein a mixture prepared by addition of a solid flavonol-containingcomposition to the aqueous solution of anthocyanin is heated aboveambient temperature to assist in dissolution of the flavonol component,cooled, and any remaining undisolved flavonol removed by filtrationand/or centrifugation.
 15. A method according to claim 1, wherein asolid flavonol-containing composition is dissolved in ethanol or anethanolic composition and added to an aqueous solution comprisinganthocyanin.
 16. A method according to claim 1, wherein aflavonol-containing composition is added to and dissolved in a wine ordealcoholised wine comprising an anthocyanin.
 17. A method according toclaim 1, wherein the flavonol-containing composition is added to anddissolved in a fruit juice or vegetable extract containing anthocyanin.18. A method according to claim 1, wherein the flavonol-containingcomposition is mixed with a juice selected from the group consisting ofgrape, cranberry, blackberry, and blackcurrant.
 19. A method ofpreparing a dry composition comprising a water-soluble flavonolcomponent, the method comprising performance of the method of claim 10,and drying the resulting solution.
 20. A method according to claim 19,performance of which produces a dry composition comprising at least 0.1%flavonol.
 21. A method according to claim 19, performance of whichproduces a dry composition comprising at least 1% flavonol.
 22. A methodaccording to claim 19, performance of which produces a dry compositioncomprising at least 5% flavonol.
 23. A method according to claim 19,performance of which produces a dry composition comprising at least 10%flavonol.
 24. A method of providing a nutritional supplement withincreased flavonol solubility, said nutritional supplement being anaqueous solution comprising dissolved flavonol at a concentration of 10mg/L to 100 mg/L, the method comprising the steps of mixing, in anyorder, water, the flavonol and an anthocyanin, and forming an aqueoussolution from the mixture, the solution comprising the flavonoldissolved at a concentration in excess of 10 mg/L, said flavonolconcentration being less than the concentration of anthocyanin.
 25. Amethod according to claim 24, wherein the solution comprises flavonoldissolved at a concentration in excess of 20 mg/L.
 26. A methodaccording to claim 24, wherein the solution comprises flavonol dissolvedat a concentration in excess of 50 mg/L.
 27. A method according to claim24, wherein the solution comprises flavonol dissolved at a concentrationin excess of 100 mg/L.
 28. A method according to claim 24, wherein theaqueous solution is essentially ethanol-free.